Abstract
We have adapted the methodology of CLIP-seq (Crosslinking-Immunoprecipitation and DNA Sequencing) to map the segments of encapsidated RNAs that contact the protein shells of virions. Results from the protocol report on the RNA sequences that contact the viral capsid.
Keywords: Protein-RNA interaction, RNA virus, Viral capsid, Virion assembly, CLIP-Seq
Background
Positive-sense RNA viruses include pathogens of all life forms. Viruses with icosahedral shapes have the viral coat proteins form a protective shell around the RNA genome (Stockley et al., 2013). In the phage MS2 and the plant-infecting Brome mosaic virus (BMV), the coat protein preferentially contacts specific RNA sequences (Ni et al., 2013; Hoover et al., 2016; Rolfsson et al., 2016). These contacts could regulate the timing of RNA release during infection, viral gene expression, and viral RNA replication (Hoover et al., 2016). Identification of the capsid-RNA interactions could thus provide insights into the regulations of viral infection and provide means to inhibit viral infection. With this in mind, we have developed a method to identify the capsid-RNA contacts in purified virions using a combination of UV crosslinking, RNA fragmentation, selective precipitation of the coat protein, and next-generation sequencing of the cDNAs made from RNA fragments. The protocol below was developed for BMV virions.
Materials and Reagents
Note: All solutions should be made using sterile water with resistivity of better than 18.3 MΩ and analytical grade reagents.
Equipment
Software
Procedure
Data analysis
Notes
While this protocol has been optimized for the analysis of the RNAs that bind to the BMV capsid, the protocol can be directly adapted to map the virions of interest. The key will be the availability of highly pure virions and an antibody that can efficiently precipitate the capsid protein of interest. The construction of cDNA libraries, nex-gen DNA sequencing and data analysis can be performed in facilities that specialize in genomic analysis.
Recipes
*Note: For convenience, make 20x stocks of the buffers and dilute with sterile water to 1x for use.
Acknowledgments
This protocol was established in the work supported by grant NIH 1R01AI090280 to CK. This protocol was adapted from the work of Ni et al. (2013) and Hoover et al. (2016) and unpublished results of E. Chuang. We thank A. Kao for photography used in this protocol.
References
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